Risk factors for recurrent severe anemia among previously transfused children in Uganda: An age-matched case-control study

In resource-poor settings, transfused children often experience recurrence of severe anemia (SA) following discharge from hospital. This study determined the factors associated with recurrent severe anemia (RSA) among previously transfused Ugandan children aged less than 5 years.

R E S E A R C H A R T I C L E Open Access

Risk factors for recurrent severe anemia

among previously transfused children in

Uganda: an age-matched case-control

study

Aggrey Dhabangi1* , Richard Idro2, Chandy C John3, Walter H Dzik4, Robert Opoka2, Ronald Ssenyonga5

and Michael Boele van Hensbroek6

Abstract

Background: In resource-poor settings, transfused children often experience recurrence of severe anemia (SA) following discharge from hospital This study determined the factors associated with recurrent severe anemia (RSA) among previously transfused Ugandan children aged less than 5 years

Methods: A case-control study was conducted in five hospitals in Uganda from March 2017 to September 2018

We prospectively enrolled 196 hospitalised children who had been transfused for severe anemia 2 weeks to 6 months prior to enrollment Of these, 101 children (cases) were re-admitted with a hemoglobin [Hb] level of≤6 g/

dL and required transfusion; and 95 children (age-matched controls) were admitted for other clinical illness with a

Hb > 6 g/dL Children known to have sickle cell anemia, cancer, or bleeding disorders were excluded Clinical and laboratory evaluation were done Conditional logistic regression adjusted for age, was used to determine factors associated with RSA

Results: The median time (IQR) between the earlier transfusion and enrollment was 3.5 (1.9–5.7) months for cases, and was 5.0 (2.9–6.0) months for controls (p-value = 0.015) Risk factors (adjusted odds ratio, 95% confidence interval, and significance) for development of RSA were: hemoglobinuria (36.33, 2.19–600.66, p = 0.012); sickle cell anemia – newly diagnosed (20.26, 2.33–176.37, p = 0.006); history of earlier previous transfusions (6.95, 1.36–35.61, p = 0.020) and malaria infection (6.47, 1.17–35.70, p = 0.032)

Conclusion: Malaria chemoprevention, follow up visit for Hb check after discharge from hospital and sickle cell screening among previously transfused children represent practical strategies to prevent and identify children at risk for recurrent severe anemia The cause of hemoglobinuria in children merits further investigations

Keywords: Recurrent severe anemia, Children, Transfusion, Malaria, Hemoglobinuria, Sickle cell anemia

Background

Recent studies have indicated that up to 10% of children

who receive blood transfusion for severe anemia (SA)

return to health facilities with recurrence of severe anemia

within three months of discharge, while others die at home

[1] Moreover, children with recurrent severe anemia (RSA)

are 10 times more likely to die compared to their

non-anemic counterparts during the post-discharge period [1] However, the risk factors associated with recurrent severe anemia among previously transfused children have not been sufficiently studied

Malaria infection in the immediate post-discharge period has been identified to be a major contributing factor to RSA in children [2] Other documented risk factors include; poor socio-economic status, large family size, history of recurrent transfusions and human immuno-deficiency virus (HIV) infection [1] Recurrent life-threatening anemia in children in sub-Saharan Africa may have several underlying

* Correspondence: adhabangi@gmail.com

1 Child Health and Development Centre, Makerere University College of

Health Sciences, Mulago upper hill road, P O Box, 6717 Kampala, Uganda

Full list of author information is available at the end of the article

© The Author(s) 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

mechanisms From a pathophysiological view point, the

relative roles of insufficient erythroid production and

increased red cell destruction have not been fully defined

[3,4]

A better understanding of the risk factors for

develop-ment of RSA can identify children at highest risk and

can enhance the benefits of blood transfusion in children

[5,6] We conducted an age-matched case-control study

to determine the factors associated with recurrent severe

anemia among previously transfused Ugandan children

aged less than 5 years

Methods

Study design

This was an age-matched study with a ratio of 1:1

be-tween cases and controls

Study setting

The study was conducted at Jinja, Masaka, Hoima,

Mubende and Kamuli hospitals, in Uganda The first four

are all public regional referral hospitals serving east-central,

south-central, north-central, and western sub-regions of

Uganda respectively, while Kamuli mission hospital is a

pri-vate not-for-profit hospital located in eastern Uganda Each

of the hospitals has a pediatric ward with an in-patient bed

capacity ranging from 30 to 80

Study population, inclusion and exclusion

We enrolled children aged 2 months to 5 years with prior

severe anemia (cases and controls) that required a blood

transfusion, which was given > 2 weeks but < 6 months

prior to enrollment in the present study Cases were

defined as children who at the time of study enrollment

were re-admitted to the hospital with a hemoglobin level of

≤6 g/dL and required blood transfusion Controls were

de-fined as children who at the time of study enrollment were

being seen for other clinical illness as an inpatient or

out-patient, and had a hemoglobin level of > 6 g/dL Cases and

controls were matched using an age range of ±12 months

Children known to have sickle cell anemia (SCA), cancer,

bleeding disorders or whose anemia was caused by trauma,

were excluded

Sample size and sampling

The sample size of 196 was estimated using Open EPI

calculator; using malaria as the main risk factor with a

prevalence of 29.3% among cases and 12.1% among

controls, according to a study byPhiri KS et al 2008 [1],

and considering a two-sided confidence level of 95%, an

acceptable type 1 error of 5% and power of 80% A study

clinician evaluated all prospective participants for eligibility

at each study site and eligible participants were enrolled

consecutively

Study variables and data collection Clinical evaluations included the past medical history (in-cluding the number of previous transfusions, past hospitali-zations and diagnosis during these hospitalihospitali-zations), socio-family history and a detailed physical examination and an-thropometric measurements (weight, height and mid-upper arm circumference [MUAC]) Socio-demographic data collected included; sex, age in months, age and occupation

of caregiver, number of household members, number of children in the household, number of meals per day, among others A structured case-report form was used to record study variables

Laboratory measurements

A blood sample of 2 ml collected in an EDTA tube was taken (for cases - taken off at pre-transfusion) Hemoglobin was measured using a point-of-care device (Hemocue® 201, Angelholm, Sweden) ABO typing and Rhesus blood grouping was done using commercial reagents available at the hospital blood banks Complete blood count (FBC) tests were performed using the Mindray automated haematology analyzer (Shenzhen Mindray Bio-Medical electronics Co Ltd., Shenzhen, China) Sickle cell status (either Hb-AA, AS or SS) was determined by capillary hemoglobin electrophoresis assay (Sebia minicap, Evry-France) For patients returning within two months since previous transfusion, hemoglobin electrophoresis was deferred for at least two months HIV serology was tested using HIV-1/2 test strips (Alere Medical Co Ltd., Chiba, Japan) Urine analysis was done using URS-10 T reagent test strips (Zhejiang Orient Gene Biotech Co Ltd., Zhejiang, China) Malaria thick-smear was stained using field stain A and B Malaria rapid diagnostic test (RDT) was done using SD Bio-line malaria Ag P.f/Pan test strips (SD Standard diagnostics, INC, Alere Co., Korea) A reticu-locyte count (%) was performed from a fresh (within 2 h) sample, using a thin smear stained with new methylene blue, and the reticulocyte production index– RPI (corrects for the degree of anemia) was calculated using the method

ofPoorana et al [7] Some tests such as Hb-electrophoresis and reticulocyte count could not be performed among some urgent cases who presented in the night and for whom a pre-transfusion study sample was not obtained The presence of asexual forms of plasmodium species on

a thick malaria smear or a positive malaria RDT defined the diagnosis of malaria, while hemoglobinuria was defined

by both a history of passage of dark or red-colored urine, and confirmed evidence of ‘blood’ at urine dip-stick Sickle cell anemia and sickle cell trait were defined as the presence

of Hb-SS and Hb-AS, respectively Suspected bacteremia was a clinical diagnosis backed by laboratory evidence neu-trophilia on FBC Mean cell volume (MCV) < 70 fL defined microcytosis, while MUAC of ≤12.5 cm defined malnutri-tion (severe acute, and moderate acute malnutrimalnutri-tion) [8]

Data management and statistical analysis

Data were entered into EPI-DATA version 3.1 software

package (The EpiData Association, Odense, Denmark) and

analysed using STATA v14.0 (Stata, College Station, TX,

USA) We computed descriptive statistics and present,

medians (interquartile range), proportions for the

demo-graphic characteristics by case or control status

Associ-ation between categorical variables was assessed using odds

ratios and statistical significance determined using the

McNemar test Means of symmetrical continuous variables

were compared using the paired t-test The difference in

median time from earlier transfusion to enrollment was

evaluated with a Wilcoxon rank sum test 95% test-based

confidence intervals for odds ratios and p-values are

presented from conditional logistic regression adjusted for

children’s age as matching variable The stepwise backward

model building technique was followed to identify

signifi-cant factors after adjusting for factors with p values < 0.2

for consideration into the multivariable model Ap < 0.05

was considered statistically significance All p-values

presented are two sided

Results

A total of 101 cases and 95 age-matched controls enrolled

in the study were included in the analysis Kamuli study site

had slightly more cases than controls (Table1) The

base-line characteristics of cases and controls were comparible

except for the median time (IQR) from prior transfusion to

enrollment which was 3.5 (1.9–5.7) and 5.0 (2.9–6.0) months among cases and controls respectively (p-value = 0.015)

Matched bivariable analysis The factors that were independently associated with RSA are summaried in Table 2 History of earlier previous blood transfusions (in the period > 6 months), history of other previous admissions, passage of dark or red-colored urine and Artemisinin-based combined therapy (ACTs) use prior to admission on the current illness were signifi-cantly associated with recurrence of SA A diagnosis of malarial anemia at the most recent previous admission, diagnosis of malaria at the current admission, SCA, and hemoglobinuria were independently associated with RSA

In contrast, socio-economic factors such as occupation

of the caregiver, highest education level of the mother, number of children in the household, number of meals per day among others, were not associated with RSA Other diagnoses such as severe pneumonia, diarrhea, and urinary tract infections were infrequent Only one participant (a control) was HIV infected

Multivariable analysis All variables with a p-value < 0.2 at bivariable analysis (hemoglobinuria, sickle cell anemia, history of earlier previ-ous blood transfusions [in the period > 6 months], passage

of dark or red-colored urine, malaria diagnosis at current Table 1 Baseline characteristics of study participants

Total (n = 196) Cases (n = 101) Controls (n = 95) Age of child in months (matching criterion), mean (SD)* 31.3 (14.4) 32.2 (14.3) 30.3 (14.5) Sex, n (%)

Study site, n (%)

Caregiver relationship to the child, n (%)

Occupation of caregiver, n (%)

Table 2 Bivariable associations with recurrent severe anemia

Cases N = 101 Controls N = 95 *Crude odds ratio (95% CI) p-value Socio-demographics

Age in months, mean (SD) 32.2 (14.3) 30.3 (14.5)

Sex, n (%)

Occupation of caregiver, n (%)

Highest education level of mother, n (%)

Mother ’s age, n (%)

No of children in household, n (%)

Total no of people in household, n (%)

No of meals per day, n (%)

History

Passage of dark or red-colored urine, n (%)

ACTs use prior to admission, n (%)

Malarial anemia at most recent admission

History of other previous admissions

History of earlier previous transfusions

At current admission

Malaria diagnosis

admission, mother’s age, ACTs use prior to admission,

mal-arial anemia at most recent admission, history of other

pre-vious admissions, and suspected bacteremia) were entered

into a stepwise backward conditional logistic regression

model After controlling for all ten variables, we found that

hemoglobinuria, sickle cell anemia (Hb-SS), history of

earl-ier previous blood transfusions (in the period > 6 months),

and malaria diagnosis were each significantly associated

with RSA (Table3)

Relationship between hemoglobinuria and sex

There were 10 (23.3%) cases and 1 (2.6%) control female

participants compared to 28 (48.3%) cases and 4 (6.9%)

control male participants with hemoglobinuria respectively

However at logistic regression, the interaction between

hemoglobinuria and sex was not statistically significant

The odds of a participant with hemoglobinuria being male

were; AOR = 1.27(95% CI: 0.09–16.88, p-value =0.855) that

of females

Discussion

This study set out to determine the factors associated with

recurrent severe anemia among previously transfused

Ugandan children aged < 5 years re-admitted to hospital The results of this study suggest that hemoglobinuria, sickle cell anemia, a history of earlier previous transfusions (in the period > 6 months) and malaria are risk factors for recur-rent severe anemia In addition, history of other previous admissions, and Artemisinin-based combined therapy (ACTs) use prior to admission on the current illness are independently associated with RSA In contrast, mother’s age≥ 29 years and a diagnosis of malarial anemia at the most recent admission seem protective These findings are comparible to the findings of Phiri KS et al 2008 and Lackritz EM et al 1997 with regard to malaria and history

of earlier previous transfusions [1,2]

It is worth noting that 75.0% of cases had a diagnosis

of malarial anemia at the most recent prior admission – in the immediate past six months, before returning with RSA This underscores the role of malaria in the etiology of severe anemia among children in malaria endemic areas [9] Although children known or sus-pected to have SCA were excluded at enrolment, we found 15 children with sickle cell anemia These had not been diagnosed before Indeed, in such settings as this with a documented prevalence of sickle cell gene as

Table 2 Bivariable associations with recurrent severe anemia (Continued)

Cases N = 101 Controls N = 95 *Crude odds ratio (95% CI) p-value Suspected bacteremia diagnosis

Hemoglobinuria diagnosis

ǂ Sickle cell status, newly diagnosed

Sickle cell anemia (Hb-SS) 9 (14.1) 6 (12.0) 4.21 (1.03,17.13) 0.045 Reticulocyte production index

MUAC, n (%)

ABO blood grouping

MCV ¶

* OR by McNemar method, ǂ114 samples tested, † Group A = 28, B = 30, AB = 6 among 128 tested, no stat test done here

high as 17% [10, 11], children presenting with SA

deserve to be evaluated for SCA

This study has found hemoglobinuria (defined by history

of passage of dark or red-colored urine and the presence of

blood at urine dip-stick) to be associated with RSA This

syndrome whose cause is not well understood has recently

been documented to be common in the eastern region of

Uganda The syndrome has been associated with SA and

positive malaria RDT (despite having negative malaria

smears) but not with sickle cell disease or G6PD-de

ficiency [12] Although G6PD-deficiency, a sex-linked

disorder has been documented to be associated with

hemoglobinuria, the study byOlopot-Olupot P et al did

not find this to be so

Similarly, in our case-control although we did not

per-form G6PD assays, we found that the odds of a participant

with hemoglobinuria being male were not statistically

significant

The potential relationship between hemoglobinuria and

prior use of ACTs and/or other genetic factors in the

causation of RSA need to be confirmed Other reports

have shown the possible role of ACTs-related delayed

hemolytic anemia in causing RSA [13]

Although the reticulocyte production index was not

statistically lower among cases, the potential role of

insufficient erythrocyte production predisposing to RSA

cannot be excluded for reasons of the fewer numbers we

tested What also remains unknown is how malarial

anemia at the most recent admission may be protective

against RSA, yet malaria itself is implicated in causing

both SA and RSA This paradoxical finding has also

been reported byPhiri KS et al 2008

Nutritional status as measured by the MUAC and

socio-economic factors such as occupation of caregiver,

highest education level of mother, number of children in

the household, number of meals per day, among others were not associated with recurrent severe anemia Al-though this study did not have power to evaluate them, there is evidence to suggest that these factors among others may play a significant role in causing both SA and RSA [1, 14] Contrary to the findings ofPhiri KS et

al where HIV infection was associated with RSA, we found only one participant – a control to be HIV posi-tive This may be explained by the marked progress made with regard to elimination of mother to child transmission of HIV in Uganda [15]

In summary, recurrent severe anemia among previ-ously transfused children in Uganda occurs after about three months, and is related to hemoglobinuria, sickle cell anemia, history of earlier previous transfusions and malaria infections and/or re-infection

Limitation The current list of factors associated with RSA may not

be complete One uncertainty may be the role of socio-demographic factors such as occupation of caregiver, number of household members, number of meals among others that our study did not have power to evaluate Similarly, the fewer numbers tested for vari-ables such as reticulocyte production index further limits the power

Conclusions Evidence based interventions are needed to prevent and mitigate the problem of recurrent severe anemia among children The post-discharge malaria chemoprevention trial (NCT02671175) is currently testing the hypothesis that malaria is a key factor in the cause of post-discharge mortality and morbidity in children with severe anemia

Table 3 Multivariable results for factors associated with recurrent severe anemia

Variable Crude odds ratio (95% CI) p-value Adjusted odds ratio (95% CI) p-value History of earlier previous transfusion

Hemoglobinuria at admission

Sickle cell status

Sickle cell trait (Hb-AS) 1.44 (0.24,8.71) 0.692 16.10 (0.06,4766.8) 0.325 Sickle cell anemia (Hb-SS) 4.21 (1.03,17.13) 0.045 20.26 (2.33,176.37) 0.006 Malaria diagnosis at admission

The results of that trial are eagerly awaited However,

there is need to address the other risk factors for RSA;

such as screening for SCA among all children with SA

and a follow up visit between 2 weeks and 3 months for

Hb check after discharge from hospital (the best timing

for this check remains yet to be determined) Generally,

the problem of recurrent severe anemia among children

merits further investigation, including areas such as the

cause of hemoglobinuria and its potential relationship

with prior ACTs use

Abbreviations

ACTs: Artemisinin-based combined therapy; EDTA: Ethylene diamine

tetra-acetic acid; FBC: Complete blood count; G6PD- deficiency:

Glocose-6-Phosphate dehydrogenate deficiency; HIV: Human immuno-deficiency virus;

MCV: Mean cell volume; MUAC: Mid-Upper Arm Circumference; RDT: Rapid

diagnostic test; RSA: Recurrent severe anemia; SA: Severe anemia; SCA: Sickle

cell anemia

Acknowledgements

The authors wish to thank the clinicians: Joanita Nankwanga, Harriet Musene,

Mercy Ambisaho, Hope Orombi, Charity Katushabe and Joanita Kyakunzire,

who enrolled study participants We thank the laboratory staff: Geoffrey

Situma, Geoffrey Kitaka, Sadat Aliwuya, Robert Kirya, Agnes Kyomugisha,

Nicholas Byaruhanga, Juliet Kentaro and David Mpiima.

Funding

This study was funded by a grant from the Research Council of Norway,

through the Global Health and Vaccination Program (GLOBVAC), project

number 234487 GLOBVAC is part of the EDCTP2 program supported by the

European Union The Council had no role in the design of the study, in the

collection, analysis and interpretation of the data, or in preparation of the

manuscript.

Availability of data and materials

The datasets used and/or analysed during the current study are available

from the corresponding author on reasonable request.

Ethics approval and consent to participate.

Written informed parental consents were obtained from the caregivers of

study participants The study was reviewed and approved by Makerere

University Research and Ethics Committee (SOMREC) and the Uganda

National Council for Science and Technology (UNCST); REC # 2017 –098 and

HS- 4420 respectively.

Authors ’ contributions

This study was conceptualized by AD, MB.Vh and RI, while AD, MB.vH, RI, CCJ

and WHD designed it AD performed the research; AD, and RS analyzed and

interpreted the data AD, MB.vH, RI, RO, CCJ, WHD and RS wrote this

manuscript All authors read and approved the final manuscript.

Consent for publication

Not applicable.

Competing interests

The authors have no competing interests to declare.

Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations.

Author details

1 Child Health and Development Centre, Makerere University College of

Health Sciences, Mulago upper hill road, P O Box, 6717 Kampala, Uganda.

2

Department of Pediatrics and Child Health, Makerere University College of

Health Sciences, Kampala, Uganda 3 Ryan White Centre for Pediatric

Infectious Disease and Global Health, Indiana University School of Medicine,

4

University / Massachusetts General Hospital, Boston, MA, USA 5 Clinical trials unit, Department of Epidemiology and Biostatistics, School of Public Health, Makerere University College of Health Sciences, Kampala, Uganda.

6

Department of Global Child Health, Emma Children ’s Hospital, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

Received: 26 November 2018 Accepted: 9 January 2019

References

1 Phiri KS, Calis JC, Faragher B, Nkhoma E, Ng'oma K, Mangochi B, et al Long term outcome of severe anaemia in Malawian children PLoS One 2008;3(8): e2903.

2 Lackritz EM, Hightower AW, Zucker JR, Ruebush TK 2nd, Onudi CO, Steketee

RW, et al Longitudinal evaluation of severely anemic children in Kenya: the effect of transfusion on mortality and hematologic recovery AIDS 1997; 11(12):1487 –94.

3 Boele van Hensbroek M, Calis JC, Phiri KS, Vet R, Munthali F, Kraaijenhagen

R, et al Pathophysiological mechanisms of severe anaemia in Malawian children PLoS One 2010;5(9):e12589.

4 van Hensbroek MB, Jonker F, Bates I Severe acquired anaemia in Africa: new concepts Br J Haematol 2011;154(6):690 –5.

5 Dzik WH Innocent lives lost and saved: the importance of blood transfusion for children in sub-Saharan Africa BMC Med 2015;13:22.

6 Thomas J, Ayieko P, Ogero M, Gachau S, Makone B, Nyachiro W, et al Blood transfusion delay and outcome in county hospitals in Kenya Am J Trop Med Hyg 2017;96(2):511 –7.

7 Priya PP, RS A Role of absolute reticulocyte count in evaluation of pancytopenia-a hospital based study J Clin Diagn Res 2014;8(8):Fc01 –03.

8 Ministry of Health, Republic of Uganda Integrated management of acute malnutrition guidelines Ministry of Health The Republic of Uganda 2010;1:7 –14.

9 Calis JC, Phiri KS, Faragher EB, Brabin BJ, Bates I, Cuevas LE, et al Severe anemia in Malawian children N Engl J Med 2008;358(9):888 –99.

10 Ndeezi G, Kiyaga C, Hernandez AG, Munube D, Howard TA, Ssewanyana I, et

al Burden of sickle cell trait and disease in the Uganda sickle surveillance study (US3): a cross-sectional study Lancet Glob Health 2016;4(3):e195 –200.

11 Suchdev PS, Ruth LJ, Earley M, Macharia A, Williams TN The burden and consequences of inherited blood disorders among young children in western Kenya Matern Child Nutr 2014;10(1):135 –44.

12 Olupot-Olupot P, Engoru C, Uyoga S, Muhindo R, Macharia A, Kiguli S, et al High frequency of Blackwater fever among children presenting to hospital with severe febrile illnesses in eastern Uganda Clin Infect Dis 2017;64(7):939 –46.

13 Burri C, Ferrari G, Ntuku HM, Kitoto AT, Duparc S, Hugo P, et al Delayed anemia after treatment with injectable artesunate in the Democratic Republic

of the Congo: a manageable issue Am J Trop Med Hyg 2014;91(4):821 –3.

14 Moschovis PP, Wiens MO, Arlington L, Antsygina O, Hayden D, Dzik W, et al Individual, maternal and household risk factors for anaemia among young children in sub-Saharan Africa: a cross-sectional study BMJ Open 2018;8(5): e019654.

15 Bannink-Mbazzi F, Lowicki-Zucca M, Ojom L, Kabasomi SV, Esiru G, Homsy J High PMTCT program uptake and coverage of mothers, their partners, and babies in northern Uganda: achievements and lessons learned over 10 years of implementation (2002-2011) J Acquir Immune Defic Syndr 2013;62(5):e138 –45.